ANTnIcIoBIAL AGzNTS AND CnzxTAuPYr, Oct. 1977, P. 518-522 Copyright 0 1977 American Society for Microbiology
Vol. 12, No. 4 Printed in U.S.A.
Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells TOMIHIKO HIGUCHI,t* TORU NAKAMURA,' HARUTO UCHINO,1 AND GYOICHI WAKISAKA2 First Division, Department ofInternal Medicine, Faculty of Medicine, Kyoto University, Sakyo, Kyoto 606,' and Shiga College of Medicine, Otu, Shiga 520, Japan2
Received for publication 19 April 1977
Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes.
The enzymes responsible for purine nucleotide synthesis have been only partially studied in L1210 cells (4, 26). Few studies have been done on the characterization of the enzymes that are essential to the mechanism of action of 6-mercaptopurine (6-MP) in humans (5, 19, 20). It is well known that nucleotides can be formed by means of a de novo pathway from single metabolites or, alternatively, synthesized from preformed purine bases by means of salvage pathways. Synthesis of purine nucleotides from preformed purines in cells has been shown to be controlled more extensively by the endogenous supply of phosphoribosylpyrophosphate (PRPP) than by the activity of the purine phosphoribosyltransferase in experimental animal tumor cells (7), human erythrocytes (24), and lymphoblasts (6). To study the regulation of purine nucleotide synthesis and to clarify the mechanism of action of 6-MP in humans, we have undertaken comparative studies on the salvage and de novo pathways, the cellular concentration of PRPP, and the enzymes responsible for PRPP formation in human leukemic leukocytes and L1210 cells.
been given 6-MP during the preceding month. Blood was drawn by venipuncture into heparinized tubes. The plasma and buffy coat were separated by centrifugation in the cold and then removed. Sedimented erythrocytes were washed twice with cold, 0.9% NaCl solution. Isolation of leukocytes. Leukocytes were isolated from peripheral blood by the dextran sedimentation procedure (23). The cells were mixed with 1 volume of 3% dextran in 0.9% NaCl solution and allowed to sediment at room temperature for 30 to 45 min until a good separation was noted. The supernatant containing leukocytes, platelets, and some erythrocytes was decanted into a test tube in which all subsequent steps were performed. The cells were sedimented at 500 x g for 10 min, washed with 0.9% NaCl solution, and sedimented at 500 x g for 10 min. The cells were suspended in 0.9% NaCl solution and sedimented as described above. The leukocyte pellet was suspended in 1 ml of 0.9% NaCl solution. Contaminating erythrocytes were lysed by adding 3 volumes of water at 0°C. After 40 s, isotonicity was restored by adding 1 volume of 3.6% NaCl solution. The unbroken cells were collected by centrifugation at 500 x g for 10 min, washed twice with 0.9% NaCl solution, resuspended, and centrifuged. Isolation of L1210 cells. L1210 cells, harvested from mice 7 days after interperitoneal inoculation of 3 x 10" cells, were washed twice with 0.9% NaCl MATERIALS AND METHODS solution. Erythrocytes were removed by lysis with Isolation of erythrocytes. Blood was obtained hypotonic NaCl solution as described above. from patients with leukemia, none of whom had Determination of thioinosinic acid (TIMP) formation. The leukocytes or L1210 cells were sust Present address: Department of Internal Medicine, pended in Eagle minimum essential medium conShiga Center for Adult Diseases, Moriyama, Shiga 524, taining 3G% calf serum in a concentration of 3 x 107 Japan. cells per ml. The cell suspension (final volume, 10 518
VOL. 12, 1977
6-MERCAPTOPURINE METABOLISM IN LEUKEMIC CELLS
ml) was incubated at 37°C for 60 min in the presence of 0.05 -,umol of 6[8-14C]MP. At the end of the incubation the mixture was chilled in an ice bath, and the cells were collected by centrifugation and washed twice with 1 ml of Eagle minimum essential medium. The acid-soluble fraction was obtained by adding 0.05 ml of 10 M perchloric acid and then centrifuging. The residue was washed again with 1 ml of 0.5 M perchloric acid. The extracts were combined and neutralized with potassium hydroxide. After removal of insoluble perchloric acid, the neutralized extracts were chromatographed on a column of Dowex-1 formate resin (1 by 20 cm) with the appropriate carriers, with a formic acid gradient elution system (0 to 20 N). Column eluates were collected in 5-ml fractions with an automatic fraction collector. Nucleotides formed were determined by measuring the radioactivity of 0.5 ml of each fraction in toluene-based scintillation fluid by a Packard Tri-Carb scintillation counter (Packard Instrument Co., Inc., Rockville, Md.). They are presented as nanomoles formed per 3 x 107 cells per h. Compared with the radioactivity of nucleotides in both human leukemic leukocytes and L1210 cells, little radioactivity was detected in the nucleoside region. Extraction of PRPP. The sedimented leukocytes or L1210 cells, prepared as described above, were suspended in 1 ml of ethylenediaminetetraacetic acid (EDTA), pH 7.4, at a concentration of 2 x 107 cells per ml. The suspensions were deproteinized by heating in boiling water for 2 min and then promptly chilling in an ice bath. The denatured protein was sedimented by centrifugation, and the clear, supernatant fluid was used immediately for assaying the PRPP content. The recovery test showed that EDTA substantially prevented decomposition of PRPP (
Vol. 12, No. 4 Printed in U.S.A.
Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells TOMIHIKO HIGUCHI,t* TORU NAKAMURA,' HARUTO UCHINO,1 AND GYOICHI WAKISAKA2 First Division, Department ofInternal Medicine, Faculty of Medicine, Kyoto University, Sakyo, Kyoto 606,' and Shiga College of Medicine, Otu, Shiga 520, Japan2
Received for publication 19 April 1977
Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate of formation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes.
The enzymes responsible for purine nucleotide synthesis have been only partially studied in L1210 cells (4, 26). Few studies have been done on the characterization of the enzymes that are essential to the mechanism of action of 6-mercaptopurine (6-MP) in humans (5, 19, 20). It is well known that nucleotides can be formed by means of a de novo pathway from single metabolites or, alternatively, synthesized from preformed purine bases by means of salvage pathways. Synthesis of purine nucleotides from preformed purines in cells has been shown to be controlled more extensively by the endogenous supply of phosphoribosylpyrophosphate (PRPP) than by the activity of the purine phosphoribosyltransferase in experimental animal tumor cells (7), human erythrocytes (24), and lymphoblasts (6). To study the regulation of purine nucleotide synthesis and to clarify the mechanism of action of 6-MP in humans, we have undertaken comparative studies on the salvage and de novo pathways, the cellular concentration of PRPP, and the enzymes responsible for PRPP formation in human leukemic leukocytes and L1210 cells.
been given 6-MP during the preceding month. Blood was drawn by venipuncture into heparinized tubes. The plasma and buffy coat were separated by centrifugation in the cold and then removed. Sedimented erythrocytes were washed twice with cold, 0.9% NaCl solution. Isolation of leukocytes. Leukocytes were isolated from peripheral blood by the dextran sedimentation procedure (23). The cells were mixed with 1 volume of 3% dextran in 0.9% NaCl solution and allowed to sediment at room temperature for 30 to 45 min until a good separation was noted. The supernatant containing leukocytes, platelets, and some erythrocytes was decanted into a test tube in which all subsequent steps were performed. The cells were sedimented at 500 x g for 10 min, washed with 0.9% NaCl solution, and sedimented at 500 x g for 10 min. The cells were suspended in 0.9% NaCl solution and sedimented as described above. The leukocyte pellet was suspended in 1 ml of 0.9% NaCl solution. Contaminating erythrocytes were lysed by adding 3 volumes of water at 0°C. After 40 s, isotonicity was restored by adding 1 volume of 3.6% NaCl solution. The unbroken cells were collected by centrifugation at 500 x g for 10 min, washed twice with 0.9% NaCl solution, resuspended, and centrifuged. Isolation of L1210 cells. L1210 cells, harvested from mice 7 days after interperitoneal inoculation of 3 x 10" cells, were washed twice with 0.9% NaCl MATERIALS AND METHODS solution. Erythrocytes were removed by lysis with Isolation of erythrocytes. Blood was obtained hypotonic NaCl solution as described above. from patients with leukemia, none of whom had Determination of thioinosinic acid (TIMP) formation. The leukocytes or L1210 cells were sust Present address: Department of Internal Medicine, pended in Eagle minimum essential medium conShiga Center for Adult Diseases, Moriyama, Shiga 524, taining 3G% calf serum in a concentration of 3 x 107 Japan. cells per ml. The cell suspension (final volume, 10 518
VOL. 12, 1977
6-MERCAPTOPURINE METABOLISM IN LEUKEMIC CELLS
ml) was incubated at 37°C for 60 min in the presence of 0.05 -,umol of 6[8-14C]MP. At the end of the incubation the mixture was chilled in an ice bath, and the cells were collected by centrifugation and washed twice with 1 ml of Eagle minimum essential medium. The acid-soluble fraction was obtained by adding 0.05 ml of 10 M perchloric acid and then centrifuging. The residue was washed again with 1 ml of 0.5 M perchloric acid. The extracts were combined and neutralized with potassium hydroxide. After removal of insoluble perchloric acid, the neutralized extracts were chromatographed on a column of Dowex-1 formate resin (1 by 20 cm) with the appropriate carriers, with a formic acid gradient elution system (0 to 20 N). Column eluates were collected in 5-ml fractions with an automatic fraction collector. Nucleotides formed were determined by measuring the radioactivity of 0.5 ml of each fraction in toluene-based scintillation fluid by a Packard Tri-Carb scintillation counter (Packard Instrument Co., Inc., Rockville, Md.). They are presented as nanomoles formed per 3 x 107 cells per h. Compared with the radioactivity of nucleotides in both human leukemic leukocytes and L1210 cells, little radioactivity was detected in the nucleoside region. Extraction of PRPP. The sedimented leukocytes or L1210 cells, prepared as described above, were suspended in 1 ml of ethylenediaminetetraacetic acid (EDTA), pH 7.4, at a concentration of 2 x 107 cells per ml. The suspensions were deproteinized by heating in boiling water for 2 min and then promptly chilling in an ice bath. The denatured protein was sedimented by centrifugation, and the clear, supernatant fluid was used immediately for assaying the PRPP content. The recovery test showed that EDTA substantially prevented decomposition of PRPP (
